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1.
J Allergy Clin Immunol ; 144(3): 825-838, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30926529

RESUMO

BACKGROUND: Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients. OBJECTIVE: We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction. METHODS: We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis. RESULTS: We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up. CONCLUSIONS: Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.

2.
J Allergy Clin Immunol ; 142(4): 1272-1284, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29421274

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. OBJECTIVE: To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. METHODS: Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. RESULTS: CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. CONCLUSION: Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity.


Assuntos
Plaquetas/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Adolescente , Adulto , Animais , Autoimunidade , Ligante de CD40/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Inflamação/sangue , Inflamação/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contagem de Plaquetas , Síndrome de Wiskott-Aldrich/sangue , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Adulto Jovem
3.
J Allergy Clin Immunol ; 142(3): 928-941.e8, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29241731

RESUMO

BACKGROUND: Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene (RAG) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor. OBJECTIVE: We sought to determine the efficacy of lentiviral vector (LV)-mediated GT in the murine model of OS (Rag2R229Q/R229Q) in correcting immunodeficiency and autoimmunity. METHODS: Lineage-negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild-type or OS-untransduced lineage-negative cells. Immunophenotyping, T-dependent and T-independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed. RESULTS: LV-mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild-type bone marrow (BM)-transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T-cell repertoire normalized, and gene-corrected mice responded properly to challenges in vivo. Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus. CONCLUSIONS: Our data show that LV-mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.


Assuntos
Proteínas de Ligação a DNA/genética , Terapia Genética , Lentivirus/genética , Imunodeficiência Combinada Severa/terapia , Animais , Autoimunidade , Linfócitos B/imunologia , Modelos Animais de Doenças , Feminino , Inflamação/imunologia , Inflamação/terapia , Contagem de Linfócitos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologia
4.
J Leukoc Biol ; 103(3): 577-590, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28851742

RESUMO

Microthrombocytopenia is the clinical hallmark of WAS, a rare X-linked immunodeficiency that is characterized by eczema, autoimmunity, and cancer susceptibility. This disease is caused by mutations in the WAS gene, which is expressed in hematopoietic cells and regulates actin cytoskeleton remodeling thereby modulating various cellular functions, including motility, immunologic synapse assembly, and signaling. Despite extensive studies that have provided great insight into the relevance of this molecule to innate and cellular immunity, the exact mechanisms of microthrombocytopenia in WAS are still unknown. This review focuses on the recent progress made in dissecting the pathogenesis of platelet defects in patients with WAS and their murine counterparts. In parallel, we will provide an overview of the state-of-the art platelets as immune modulators at the interface between hemostasis and the immune system, which suggests that these cells may have a direct role in the pathogenesis of immune dysregulation in WAS.


Assuntos
Autoimunidade , Plaquetas/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Animais , Plaquetas/patologia , Humanos , Transdução de Sinais , Síndrome de Wiskott-Aldrich/patologia
5.
Front Immunol ; 8: 490, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28512459

RESUMO

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by mutations in the gene encoding the hematopoietic-specific WAS protein (WASp). WAS is frequently associated with autoimmunity, indicating a critical role of WASp in maintenance of tolerance. The role of B cells in the induction of autoreactive immune responses in WAS has been investigated in several settings, but the mechanisms leading to the development of autoimmune manifestations have been difficult to evaluate in the mouse models of the disease that do not spontaneously develop autoimmunity. We performed an extensive characterization of Was-/- mice that provided evidence of the potential alteration in B cell selection, because of the presence of autoantibodies against double-stranded DNA, platelets, and tissue antigens. To uncover the mechanisms leading to the activation of the potentially autoreactive B cells in Was-/- mice, we performed in vivo chronic stimulations with toll-like receptors agonists (LPS and CpG) and apoptotic cells or infection with lymphocytic choriomeningitis virus. All treatments led to increased production of autoantibodies, increased proteinuria, and kidney tissue damage in Was-/- mice. These findings demonstrate that a lower clearance of pathogens and/or self-antigens and the resulting chronic inflammatory state could cause B cell tolerance breakdown leading to autoimmunity in WAS.

6.
J Exp Med ; 213(3): 355-75, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26926994

RESUMO

Omenn syndrome (OS) is caused by hypomorphic Rag mutations and characterized by a profound immunodeficiency associated with autoimmune-like manifestations. Both in humans and mice, OS is mediated by oligoclonal activated T and B cells. The role of microbial signals in disease pathogenesis is debated. Here, we show that Rag2(R229Q) knock-in mice developed an inflammatory bowel disease affecting both the small bowel and colon. Lymphocytes were sufficient for disease induction, as intestinal CD4 T cells with a Th1/Th17 phenotype reproduced the pathological picture when transplanted into immunocompromised hosts. Moreover, oral tolerance was impaired in Rag2(R229Q) mice, and transfer of wild-type (WT) regulatory T cells ameliorated bowel inflammation. Mucosal immunoglobulin A (IgA) deficiency in the gut resulted in enhanced absorption of microbial products and altered composition of commensal communities. The Rag2(R229Q) microbiota further contributed to the immunopathology because its transplant into WT recipients promoted Th1/Th17 immune response. Consistently, long-term dosing of broad-spectrum antibiotics (ABXs) in Rag2(R229Q) mice ameliorated intestinal and systemic autoimmunity by diminishing the frequency of mucosal and circulating gut-tropic CCR9(+) Th1 and Th17 T cells. Remarkably, serum hyper-IgE, a hallmark of the disease, was also normalized by ABX treatment. These results indicate that intestinal microbes may play a critical role in the distinctive immune dysregulation of OS.


Assuntos
Autoimunidade , Proteínas de Ligação a DNA/metabolismo , Microbioma Gastrointestinal , Inflamação/imunologia , Inflamação/patologia , Transferência Adotiva , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Autoimunidade/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Carga Bacteriana/efeitos dos fármacos , Translocação Bacteriana/efeitos dos fármacos , Colite/imunologia , Colite/patologia , Proteínas de Ligação a DNA/deficiência , Microbioma Gastrointestinal/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Imunoglobulina E/metabolismo , Imunofenotipagem , Inflamação/microbiologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Tropismo/efeitos dos fármacos
7.
J Clin Invest ; 125(10): 3941-51, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26368308

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.


Assuntos
Linfócitos B/imunologia , Terapia Genética , Vetores Genéticos/uso terapêutico , Tolerância Imunológica , Proteína da Síndrome de Wiskott-Aldrich/uso terapêutico , Síndrome de Wiskott-Aldrich/terapia , Adulto , Sequência de Aminoácidos , Medula Óssea/patologia , Criança , Pré-Escolar , Deleção Clonal , Células Clonais/imunologia , Humanos , Lentivirus/genética , Masculino , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Recombinantes de Fusão , Linfócitos T Reguladores/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
8.
J Immunol ; 194(9): 4144-53, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25825446

RESUMO

The immune and the skeletal system are tightly interconnected, and B lymphocytes are uniquely endowed with osteo-interactive properties. In this context, receptor activator of NF-κB (RANK) ligand (RANKL) plays a pivotal role in lymphoid tissue formation and bone homeostasis. Although murine models lacking RANK or RANKL show defects in B cell number, the role of the RANKL-RANK axis on B physiology is still a matter of debate. In this study, we have characterized in detail B cell compartment in Rankl(-/-) mice, finding a relative expansion of marginal zone B cells, B1 cells, and plasma cells associated with increased Ig serum levels, spontaneous germinal center formation, and hyperresponse to CD40 triggering. Such abnormalities were associated with an increased frequency of regulatory B cells and augmented B cell-derived IL-10 production. Remarkably, in vivo IL-10-R blockade reduced T cell-triggered plasma cell differentiation and restrained the expansion of regulatory B cells. These data point to a novel role of the RANKL-RANK axis in the regulation of B cell homeostasis and highlight an unexpected link between IL-10 CD40 signaling and the RANKL pathway.


Assuntos
Linfócitos B/imunologia , Interleucina-10/imunologia , Ligante RANK/deficiência , Ligante RANK/imunologia , Animais , Camundongos , Camundongos Knockout
9.
J Allergy Clin Immunol ; 136(3): 692-702.e2, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25792466

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene-corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. OBJECTIVE: Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. METHODS: We evaluated B-cell counts, B-cell subset distribution, B cell-activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. RESULTS: After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19(+)CD21(-)CD35(-) and CD21(low) B cells and a reduction in B cell-activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. CONCLUSIONS: We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Autoanticorpos/biossíntese , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Criança , Pré-Escolar , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunoglobulinas/biossíntese , Imunofenotipagem , Lactente , Lentivirus/genética , Masculino , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução Genética , Condicionamento Pré-Transplante , Transplante Autólogo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia
10.
J Allergy Clin Immunol ; 133(3): 799-806.e10, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24506932

RESUMO

BACKGROUND: Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor. OBJECTIVE: We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function. METHODS: Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation. RESULTS: Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT. CONCLUSIONS: ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.


Assuntos
Adenosina Desaminase/deficiência , Linfócitos B/fisiologia , Terapia de Reposição de Enzimas , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Adenosina Desaminase/genética , Adenosina Desaminase/uso terapêutico , Adolescente , Fator Ativador de Células B/fisiologia , Linfócitos B/imunologia , Criança , Pré-Escolar , Humanos , Lactente
11.
J Autoimmun ; 50: 42-50, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24369837

RESUMO

Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21(low) B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients.


Assuntos
Autoimunidade , Linfócitos B/imunologia , Suscetibilidade a Doenças/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Síndrome de Wiskott-Aldrich/imunologia , Fator Ativador de Células B/sangue , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/patologia , Medula Óssea/imunologia , Medula Óssea/patologia , Diferenciação Celular , Movimento Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Expressão Gênica , Homeostase/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Memória Imunológica , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
12.
Science ; 341(6148): 1233151, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23845947

RESUMO

Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.


Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Vetores Genéticos , Humanos , Lentivirus , Masculino , Transdução Genética , Integração Viral
13.
J Exp Med ; 210(2): 355-74, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23337808

RESUMO

Mutations in Wiskott-Aldrich syndrome (WAS) protein (WASp), a regulator of actin dynamics in hematopoietic cells, cause WAS, an X-linked primary immunodeficiency characterized by recurrent infections and a marked predisposition to develop autoimmune disorders. The mechanisms that link actin alterations to the autoimmune phenotype are still poorly understood. We show that chronic activation of plasmacytoid dendritic cells (pDCs) and elevated type-I interferon (IFN) levels play a role in WAS autoimmunity. WAS patients display increased expression of type-I IFN genes and their inducible targets, alteration in pDCs numbers, and hyperresponsiveness to TLR9. Importantly, ablating IFN-I signaling in WASp null mice rescued chronic activation of conventional DCs, splenomegaly, and colitis. Using WASp-deficient mice, we demonstrated that WASp null pDCs are intrinsically more responsive to multimeric agonist of TLR9 and constitutively secrete type-I IFN but become progressively tolerant to further stimulation. By acute silencing of WASp and actin inhibitors, we show that WASp-mediated actin polymerization controls intracellular trafficking and compartmentalization of TLR9 ligands in pDCs restraining exaggerated activation of the TLR9-IFN-α pathway. Together, these data highlight the role of actin dynamics in pDC innate functions and imply the pDC-IFN-α axis as a player in the onset of autoimmune phenomena in WAS disease.


Assuntos
Actinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon Tipo I/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/antagonistas & inibidores , Animais , Autoimunidade , Sequência de Bases , Células Dendríticas/patologia , Modelos Animais de Doenças , Endocitose , Feminino , Humanos , Imunidade Inata , Interferon Tipo I/genética , Interferon-alfa/biossíntese , Interferon-alfa/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Transdução de Sinais , Receptor Toll-Like 9/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética
14.
Cardiovasc Res ; 96(3): 381-90, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22875468

RESUMO

AIMS: The suppressors of cytokine signalling (SOCS) are identified inhibitors of cytokine and growth factor signalling that act via the Janus kinase (JAK) signal transducers and activators of transcription (STAT) pathways. Aberrant JAK/STAT signalling promotes progression from hypertrophy to heart failure. Little information is available concerning the role of SOCS in the transition from hypertrophy to heart failure. To this aim, we investigated the effects of SOCS1 overexpression obtained by in vivo adeno-associated gene transfer using an aortopulmonary cross-clamping technique in a chronic pressure-overload cardiac rat model. METHODS AND RESULTS: Rats were randomized into four groups: sham-operated (n = 18), aortic banding (AB) (n = 18), AB + viral vector encoding for haemoagglutinin (AB + HA, n = 16), and AB + viral vector encoding for SOCS1 (AB + SOCS1, n = 18). Echocardiographic and haemodynamic measurements were performed 15 weeks after banding. While SOCS3 was upregulated during the hypertrophic phase, SOCS1 transcript levels increased significantly between 15 and 20 weeks. Remodelling was markedly worse in AB + SOCS1, showed larger left ventricular internal dimensions (+16%), higher end-diastolic pressures (+57%) and wall stress (+45%), and reduced fractional shortening (-32%) compared with AB + HA; apoptotic rate was increased three-fold and the gp130 pathway was inhibited. Ex vivo experiments showed that mechanical stretch upregulated SOCS1 expression, which was in turn attenuated by tumour necrosis factor-α (TNF-α) inhibition. CONCLUSION: Enhanced SOCS1 myocardial signalling is associated with accelerated transition from hypertrophy to failure in an established model of pressure overload. SOCS1 may represent an attractive target for the prevention of heart failure progression.


Assuntos
Receptor gp130 de Citocina/metabolismo , Técnicas de Transferência de Genes , Insuficiência Cardíaca/etiologia , Hipertrofia Ventricular Esquerda/etiologia , Janus Quinase 1/metabolismo , Miocárdio/enzimologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Angiopoietina-2/metabolismo , Animais , Apoptose , Dependovirus/genética , Modelos Animais de Doenças , Progressão da Doença , Vetores Genéticos , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Janus Quinase 1/genética , Masculino , Miocárdio/patologia , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Transcrição STAT3/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de Tempo , Ultrassonografia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular Esquerda
15.
Front Immunol ; 3: 209, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22826711

RESUMO

Wiskott-Aldrich Syndrome (WAS) is a severe X-linked Primary Immunodeficiency that affects 1-10 out of 1 million male individuals. WAS is caused by mutations in the WAS Protein (WASP) expressing gene that leads to the absent or reduced expression of the protein. WASP is a cytoplasmic protein that regulates the formation of actin filaments in hematopoietic cells. WASP deficiency causes many immune cell defects both in humans and in the WAS murine model, the Was(-/-) mouse. Both cellular and humoral immune defects in WAS patients contribute to the onset of severe clinical manifestations, in particular microthrombocytopenia, eczema, recurrent infections, and a high susceptibility to develop autoimmunity and malignancies. Autoimmune diseases affect from 22 to 72% of WAS patients and the most common manifestation is autoimmune hemolytic anemia, followed by vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA nephropathy. Many groups have widely explored immune cell functionality in WAS partially explaining how cellular defects may lead to pathology. However, the mechanisms underlying the occurrence of autoimmune manifestations have not been clearly described yet. In the present review, we report the most recent progresses in the study of immune cell function in WAS that have started to unveil the mechanisms contributing to autoimmune complications in WAS patients.

16.
J Allergy Clin Immunol ; 127(6): 1376-84.e5, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21531013

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety. OBJECTIVE: To evaluate B-cell reconstitution and correction of B-cell phenotype in GT-treated mice. METHODS: We transplanted Was(-/-) mice sublethally irradiated (700 rads) with lineage marker-depleted bone marrow wild-type cells, Was(-/-) cells untransduced or transduced with the w1.6W lentiviral vector and analyzed B-cell reconstitution in bone marrow, spleen, and peritoneum. RESULTS: Here we show that WAS protein(+) B cells were present in central and peripheral B-cell compartments from GT-treated mice and displayed the strongest selective advantage in the splenic marginal zone and peritoneal B1 cell subsets. After GT, splenic architecture was improved and B-cell functions were restored, as demonstrated by the improved antibody response to pneumococcal antigens and the reduction of serum IgG autoantibodies. CONCLUSION: WAS GT leads to improvement of B-cell functions, even in the presence of a mixed chimerism, further validating the clinical application of the w1.6W lentiviral vector.


Assuntos
Linfócitos B/imunologia , Terapia Genética/métodos , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/terapia , Animais , Antígenos T-Independentes/administração & dosagem , Autoanticorpos/sangue , Linfócitos B/metabolismo , Transplante de Medula Óssea , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
17.
J Allergy Clin Immunol ; 125(2): 439-448.e8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20159256

RESUMO

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. OBJECTIVE: The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. METHODS: A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. RESULTS: The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. CONCLUSION: Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS.


Assuntos
Tecido Linfoide/imunologia , Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Adulto , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Separação Celular , Análise Mutacional de DNA , Citometria de Fluxo , Humanos , Tecido Linfoide/citologia , Masculino , Microscopia Confocal , Dados de Sequência Molecular , Mosaicismo , Mutação , Reação em Cadeia da Polimerase
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